Lipoprotein associated phospholipase A2, inhibitors thereof and use of the same in diagnosis and therapy

ABSTRACT

The enzyme Lp-PLA 2  in purified form, an isolated nucleic acid molecule encoding Lp-PLA 2 , the use of an inhibitor of the enzyme Lp-PLA 2  in therapy and a method of screening compounds to identify those compounds.

INTRODUCTION

This application is a continuation of U.S. patent application Ser. No.10/173,233, filed Jun. 14, 2002, now issued as U.S. Pat. No. 7,052,862,which is a continuation of U.S. patent application Ser. No. 09/569,899filed May 12, 2000, pending, which is a continuation of U.S. patentapplication Ser. No. 09/294,384 filed Apr. 20, 1999, issued as U.S. Pat.No. 6,177,257, which is a continuation of U.S. patent application Ser.No. 08/387,858 filed Feb. 24, 1995, issued as U.S. Pat. No. 5,981,252,which was the National Stage of International Application No.PCT/GB94/01374, filed Jun. 24, 1994, which claims benefit of priority toGB9400413.2, filed Jan. 11, 1994 and GB9313144.9 filed Jun. 25, 1993,each of which are herein incorporated by reference in their entirety.

The present invention relates to the use of inhibitors of an enzyme inthe therapy, in particular in the treatment of atherosclerosis. Thepresent invention also relates to the isolation and purification of theenzyme, to isolated nucleic acids encoding the enzyme, to recombinanthost cells transformed with DNA encoding the enzyme, to the use of theenzyme in diagnosing a patient's susceptibility to atherosclerosis, andto the use of the enzyme in identifying compounds which are potentiallyuseful for the treatment of atherosclerosis.

Lipoprotein Associated Phospholipase A₂ (Lp-PLA₂), also previously knownin the art as Platelet Activating Factor Acetyl Hydrolase (PAF acetylhydrolase). During the conversion of LDL to its oxidised form, Lp-PLA₂is responsible for hydrolysing the sn-2 ester of oxidatively modifiedphosphatidylcholine to give lyso-phosphatidylcholine and an oxidativelymodified fatty acid. Both of these products of Lp-PLA₂ action are potentchemoattractants for circulating monocytes. As such, this enzyme isthought to be responsible for the accumulation of cells loaded withcholesterol ester in the arteries, causing the characteristic ‘fattystreak’ associated with the early stages of atherosclerosis. Inhibitionof the Lp-PLA₂ enzyme would therefore be expected to stop the build upof this fatty streak (by inhibition of the formation oflysophosphatidylcholine), and so be useful in the treatment ofatherosclerosis. In addition, it is proposed that Lp-PLA₂ plays a directrole in LDL oxidation. This is due to the poly unsaturated fattyacid-derived lipid peroxide products of Lp-PLA₂ action contributing toand enhancing the overall oxidative process. In keeping with this idea,Lp-PLA₂ inhibitors inhibit LDL oxidation. Lp-PLA₂ inhibitors maytherefore have a general application in any disorder that involves lipidperoxidation in conjunction with the enzyme activity, for example inaddition to conditions such as atherosclerosis and diabetes otherconditions such as rheumatoid arthritis, stroke, myocardial infarction,reperfusion injury and acute and chronic inflammation.

The present invention therefore provides in a first aspect an inhibitorof the enzyme lipoprotein associated Lp-PLA₂ for use in therapy, inparticular in the treatment of atherosclerosis. Suitable compounds ableto inhibit the Lp-PLA₂ enzyme are known in the art and include forexample, the following compounds of structure (I):

in which R is C₁₋₆alkylCONR²;

-   -   R² is hydrogen or C₁₋₆ alkyl;    -   X is oxygen, sulphur or —O(CO)—;    -   R¹ is C₈₋₂₀ alkyl;    -   Z is N(R³)₂, ^(⊕)N(R³)₃, SR³, ^(⊕)S(R³)₂, in which each group R³        is the same or different and is C₁₋₆ alkyl, OR², C₁₋₄ alkanoyl,        imidazolyl or N-methylimidazolyl    -   Suitably R² is hydrogen or C₁₋₆ alkyl; preferably R² is        hydrogen.    -   Suitably X is oxygen, sulphur or —(CO); preferably X is oxygen    -   Suitably R¹ is C₈₋₂₀ alkyl; preferably R¹ is C₁₆₋₁₈ alkyl    -   Suitably Z is N(R³)₂, ^(⊕)N(R³)₃, SR³, ^(⊕)S(R³)₂, in which each        group R³ is the same or different and is C₁₋₆ alkyl, OR², C₁₋₄        alkanoyl imidazolyl or N-methylimidazolyl; preferably Z is SR³        in which R³ is methyl or OR² in which R² is hydrogen

The compounds of structure (I) can be prepared by processes known tothose skilled in the art, for example as described in J Chem Soc ChemComn., 1993, 70–72; J Org Chem, 1983, 48, 1197 and Chem Phys Lipids,1984,35,29–37 or procedures analogous thereto.

When used in therapy, the compounds of structure (I) are formulated inaccordance with standard pharmaceutical practice.

The compounds of structure (I) and their pharmaceutically acceptablesalts which are active when given orally can be formulated as liquids,for example syrups, suspensions or emulsions, tablets, capsules and,lozenges.

A liquid formulation will generally consist of a suspension or solutionof the compound or pharmaceutically acceptable salt in a suitable liquidcarrier(s) for example, ethanol, glycerine, non-aqueous solvent, forexample polyethylene glycol oils, or water with a suspending agent,preservative, flavouring or colouring agent

A composition in the form of a tablet can be prepared using any suitablepharmaceutical carrier(s) routinely used for preparing solidformulations. Examples of such carriers include magnesium stearate,starch, lactose, sucrose and cellulose.

A composition in the form of a capsule can be prepared using routineencapsulation procedures. For example, pellets containing the activeingredient can be prepared using standard carriers and then filled intoa hard gelatin capsule; alternatively, a dispersion or suspension can beprepared using any suitable pharmaceutical carrier(s), for exampleaqueous gums, celluloses, silicates or oils and the dispersion orsuspension then filled into a soft gelatin capsule.

Typical parenteral compositions consist of a solution or suspension ofthe compound or pharmaceutically acceptable salt in a sterile aqueouscarrier or parenterally acceptable oil, for example polyethylene glycol,polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil.Alternatively, the solution can be lyophilised and then reconstitutedwith a suitable solvent just prior to administration.

A typical suppository formulation comprises a compound of formula (I) ora pharmaceutically acceptable salt thereof which is active whenadministered in this way, with a binding and/or lubricating agent suchas polymeric glycols, gelatins or cocoa butter or other low-meltingvegetable or synthetic waxes or fats.

Preferably the composition is in unit dose form such as a tablet orcapsule.

Each dosage unit for oral administration contains preferably from 1 to250 mg (and for parenteral administration contains preferably from 0.1to 25 mg) of a compound of the formula (I) or a pharmaceuticallyacceptable salt thereof calculated as the free base.

The daily dosage regimen for an adult patient may be, for example, anoral dose of between 1 mg and 500 mg, preferably-between 1 mg and 250mg, or an intravenous, subcutaneous, or intramuscular dose of between0.1 mg and 100 mg, preferably between 0.1 mg and 25 mg, of the compoundof the formula (I) or a pharmaceutically acceptable salt thereofcalculated as the free base, the compound being administered 1 to 4times per day. Suitably the compounds will be administered for a periodof continuous therapy.

The enzyme, lipoprotein associated Lp-PLA₂ has not hitherto beenavailable in isolated purified form. The present invention thereforeprovides in a further aspect, the enzyme lipoprotein associated Lp-PLA₂in purified form. By purified form is meant at least 80%, morepreferably 90%, still more preferably 95% and most preferably 99% purewith respect to other protein contaminants.

The enzyme Lp-PLA₂ may be characterised by one or more partial peptidesequences selected from SEQ ID NOS:1, 2, 3, 4, 10 and 11 or by thepartial peptide sequence comprising residues 271 to 441 or consisting ofresidues 1 to 441 of SEQ ID NO: 9. The enzyme Lp-PLA₂ may further oralternatively characterised by its molecular weight found to be 45 kDa,at least 45 kDa, 45–47 kDa, 46–47 kDa or 45–50 kDa

The invention also provides fragments of the enzyme having Lp-PLA₂activity.

The enzyme can be isolated and purified using the methods hereafterdescribed. Once isolated, the protein sequence of the enzyme can beobtained using standard techniques. In identifying said sequence, anumber of protein fragments have been identified, each of whichcomprises part of the whole sequence of the enzyme. These sequences arethemselves novel and form a further aspect of the invention.

This invention also provides isolated nucleic acid molecules encodingthe enzyme, including mRNAs, DNAs, cDNAs as well as antisense analogsthereof and biologically active and diagnostically or therapeuticallyuseful fragments thereof.

In particular, the invention provides an isolated nucleic acid moleculeconsisting of bases 1 to 1361 or 38 to 1361 or comprising the sequencecorresponding to bases 848 to 1361 of SEQ ID NO: 9.

This invention also provides recombinant vectors, such as cloning andexpression plasmids useful as reagents in the recombinant production ofthe enzyme, as well as recombinant prokaryotic and/or eukaryotic hostcells comprising the novel nucleic acid sequence.

This invention also provides nucleic acid probes comprising nucleic acidmolecules of sufficient length to specifically hybridize to the novelnucleic acid sequences.

This invention also provides an antisense oligonucleotide having asequence capable of binding with mRNAs encoding the enzyme so as toprevent the translation of said mRNA.

This invention also provides transgenic non-human animals comprising anucleic acid molecule encoding the enzyme. Also provided are methods foruse of said transgenic animals as models for mutation and SAR(structure/activity relationship) evaluation as well as in drug screens.

This invention further provides a method of screening compounds toidentify those compounds which inhibit the enzyme comprising contactingisolated enzyme with a test compound and measuring the rate of turnoverof an enzyme substrate as compared with the rate of turnover in theabsence of test compound.

“Recombinant” polypeptides refer to polypeptides produced by recombinantDNA techniques, i.e., produced from cells transformed by an exogenousDNA construct encoding the desired polypeptide. “Synthetic” polypeptidesare those prepared by chemical synthesis.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo: i.e,capable of replication under its own control

A “vector” is a replicon, such as a plasmid, phage, or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A “double-stranded DNA molecule” refers to the polymeric form ofdeoxyribonucleotides (bases adenine, guanine, thymine, or cytosine) in adouble-stranded helix, both relaxed and supercoiled. This term refersonly to the primary and secondary structure of the molecule, and doesnot limit it to any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear DNA molecules (e g,restriction. fragments), viruses, plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along the sensestrand of DNA.

A DNA “coding sequence of” or a “nucleotide sequence encoding” aparticular protein, is a DNA sequence which is transcribed andtranslated into a polypeptide when placed under the control ofappropriate regulatory sequences.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. Within the promoter sequence will be found atranscription initiation site (conveniently defined by mapping withnuclease S1), as well as protein binding domains (consensus sequences)responsible for the binding of RNA polymerase. Eukaryotic promoters willoften, but not always, contain “TATA” boxes and “CAT” boxes.

DNA “control sequences” refers collectively to promoter sequences,ribosome binding sites, polyadenylation signals, transcriptiontermination sequences, upstream regulatory domains, enhancers, and thelike, which collectively provide for the expression (i.e., thetranscription and translation) of a coding sequence in a host cell.

A control sequence “directs the expression” of a coding sequence in acell when RNA polymerase will bind the promoter sequence and transcribethe coding sequence into mRNA, which is then translated into thepolypeptide encoded by the coding sequence.

A “host cell” is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous DNA sequence.

A cell has been “transformed” by exogenous DNA when such exogenous DNAhas been introduced inside the cell membrane. Exogenous DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes and yeasts, for example, theexogenous DNA may be maintained on an episomal element, such as aplasmid. With respect to eukaryotic cells, a stably transformed ortransfected cell is one in which the exogenous DNA has become integratedinto the chromosome so that it is inherited by daughter cells throughchromosome replication. This stability is demonstrated by the ability ofthe eukaryotic cell to establish cell lines or clones comprised of apopulation of daughter cell containing the exogenous DNA.

A “clone” is a population of cells derived from a single cell or commonancestor by mitosis. A “cell line” is a clone of a primary cell that iscapable of stable growth in vitro for many generations.

Two DNA or polypeptide sequences are “substantially homologous” or“substantially the same” when at least about 85% (preferably at leastabout 90%, and most preferably at least about 95%) of the nucleotides oramino acids match over a defined length of the molecule and includesallelic variations. As used herein, substantially homologous also refersto sequences showing identity to the specified DNA or polypeptidesequence. DNA sequences that are substantially homologous can beidentified in a Southern hybridization experiment under, for example,stringent conditions, as defined for that particular system. Definingappropriate hybridization conditions is within the skill of the art. Seee.g., A “Current Protocols in Mol. Biol.” Vol. I & II, WileyInterscience. Ausbel et al. (ed.) (1992). Protein sequences that aresubstantially the same can be identified by proteolytic digestion, gelelectrophoresis and microsequencing.

The term “functionally equivalent” intends that the amino acid sequenceof the subject protein is one that will exhibit enzymatic activity ofthe same kind as that of Lp-PLA₂.

A “heterologous” region of a DNA construct is an identifiable segment ofDNA within or attached to another DNA molecule that is not found inassociation with the other molecule in nature.

This invention provides an isolated nucleic acid molecule encoding theenzyme Lp-PLA₂. One means for isolating the coding nucleic acid is. toprobe a human genomic or cDNA library with a natural or artificiallydesigned probe using art recognized procedures (See for example:“Current Protocols in Molecular Biology”, Ausubel, F. M., et al. (eds.)Greene Publishing Assoc and John Wiley Interscience, N.Y., 1989, 1992);for example using the protein fragment information disclosed herein. Theenzyme of this invention may be made by recombinant genetic engineeringtechniques. The isolated nucleic acids particularly the DNAs can beintroduced into expression vectors by operatively linking the DNA to thenecessary expression control regions (e.g. regulatory regions) requiredfor gene expression. The vectors can be introduced into the appropriatehost cells such as prokaryotic (e.g., bacterial), or eukaryotic (e.g.yeast, insect or mammalian) cells by methods well known in the art(Ausubel et al., supra). The coding sequences for the desired proteinshaving been prepared or isolated, can be cloned into a suitable vectoror replicon. Numerous cloning vectors are known to those of skill in theart, and the selection of an appropriate cloning vector is a matter ofchoice. Examples of recombinant DNA vectors for cloning and host cellswhich they can transform include the bacteriophage λ (E. coli), pBR322(E. coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106(gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290(non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillussubtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces),YIp5 (Saccharomyces), a baculovirus insect cell system, , YCp19(Saccharomyces). See generally “DNA Cloning”: Vols. I & II, Glover etal. ed IRL Press Oxford (1985) (1987) and; T. Maniatis et. al.(“Molecular Cloning” Cold Spring Harbor Laboratory (1982).

The gene can be placed under the control of a promoter, ribosome bindingsite (for bacterial expression) and, optionally, an operator(collectively referred to herein as “control” elements), so that the DNAsequence encoding the desire protein is transcribed into RNA in the hostcell transformed by a vector containing this expression construction.The coding sequence may or may not contain a signal peptide or leadersequence. The protein sequences of the present invention can beexpressed using, for example, the E. coli tac promoter or the protein Agene (spa) promoter and signal sequence. Leader sequences can be removedby the bacterial host in post-translational processing. See. e.g. U.S.Pat. Nos. 4,431,739; 4,425,437; 4,338,397.

In addition to control sequences, it may be desirable to add regulatorysequences which allow for regulation of the expression of the proteinsequences relative to the growth of the host cell. Regulatory sequencesare known to those of skill in the art, and examples include those whichcause the expression of a gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound Other types of regulatory elements may also be present in thevector, for example, enhancer sequences.

An expression vector is constructed so that the particular codingsequence is located in the vector with the appropriate regulatorysequences, the positioning and orientation of the coding sequence withrespect to the control sequences being such that the coding sequence istranscribed under the “control” of the control sequences (i.e., RNApolymerase which binds to the DNA molecule at the control sequencestranscribes the coding sequence). Modification of the coding sequencesmay be desirable to achieve this end. For example, in some cases it maybe necessary to modify the sequence so that it may be attached to thecontrol sequences with the appropriate orientation; i.e., to maintainthe reading frame. The control sequences and other regulatory sequencesmay be ligated to the coding sequence prior to insertion into a vector,such as the cloning vectors described above. Alternatively, the codingsequence can be cloned directly into an expression vector which alreadycontains the control sequences and an appropriate restriction site.Modification of the coding sequences may also be performed to altercodon usage to suit the chosen host cell, for enhanced expression

In some cases, it may be desirable to add sequences which cause thesecretion of the polypeptide from the host organism, with subsequentcleavage of the secretory signal. It may also be desirable to producemutants or analogs of the enzyme of interest. Mutants or analogs may beprepared by the deletion of a portion of the sequence encoding theprotein, by insertion of a sequence, and/or by substitution of one ormore nucleotides within the sequence. Techniques for modifyingnucleotide sequences, such as site-directed mutagenesis, are well knownto those skilled in the art. See e.g., T. Maniatis et al., supra; DNACloning. Vols. I and II, supra; Nucleic Acid Hybridization, supra.

A number of prokaryotic expression vectors are known in the art. See.e.g., U.S. Pat. Nos. 4,578,355; 4,440,859; 4,436,815; 4,431,740;4,431,739; 4,428,941; 4,425,437; 4,418,149; 4,411,994; 4,366,246;4,342,832; see also U.K. Patent Applications GB 2,121,054; GB 2,008,123;GB 2,007,675; and European Patent Application 103,395. Yeast expressionvectors are also known in the art See. e.g. U.S. Pat. Nos. 4,446,235;4,443,539; 4,430,428; see also European Patent Applications 103,409;100,561; 96,491. pSV2neo (as described in J. Mol. Appl. Genet.1:327–341) which uses the SV40 late promoter to drive expression inmammalian cells or pCDNA1 neo, a vector derived from pCDNA1 (Mol. CellBiol. 7:4125–29) which uses the CMV promoter to drive expression. Boththese latter two vectors can be employed for transient or stable(usingG418 resistance) expression in mammalian cells. Insect cell expressionsystems, e.g., Drosophila, are also useful, see for example, PCTapplications U.S. Ser. No. 89/05155 and U.S. Ser. No. 91/06838 as wellas EP application 88/3040933.

Depending on the expression system and host selected, the enzyme of thepresent invention may be produced by growing host cells transformed byan expression vector described above under conditions whereby theprotein of interest is expressed. The protein is then isolated from thehost cells and purified. If the expression system secretes the proteininto growth media, the protein can be purified directly from the media.If the protein is not secreted, it is isolated from cell lysates orrecovered from the cell membrane fraction. Where the protein islocalized to the cell surface, whole cells or isolated membranes can beused as an assayable source of the desired gene product Proteinexpressed bacterial hosts such as E. coli may require isolation frominclusion bodies and refolding. The selection of the appropriate growthconditions and recovery methods are within the skill of the art.

The identification of this novel target for the treatment ofatherosclerosis, also leads to a novel diagnostic method to diagnose apatient's susceptibility to developing atherosclerotic disease.

The present invention therefore provides in a still further aspect adiagnostic method comprising isolating a sample of blood from thepatient, and assaying said sample for Lp-PLA₂ activity. Patients thatare susceptible to atherosclerotic disease are expected to have elevatedlevels of the Lp-PLA₂ enzyme, and hence the levels of Lp-PLA₂ providesan indication of the patients susceptibility to atherosclerotic disease.Moreover, Lp-PLA₂ is found located predominantly on dense subfraction(s)of LDL which are known to be very atherogenic. Plasma Lp-PLA₂ levelscould therefore provide a ready measure of these very atherogenic smalldense LDL particles.

It is expected that the presence of the enzyme in the blood sample ofthe patient can be assayed by analysis of enzyme activity (i.e. by anassay set up against the purified enzyme as standard); or alternativelyby assaying protein content of the sample by using polyclonal ormonoclonal antibodies prepared against the purified enzyme. Monoclonal(and polyclonal) antibodies raised against the purified enzyme orfragments thereof are themselves novel and form a further aspect of theinvention.

DATA AND EXAMPLES

1. Screen for Lp-PLA₂ Inhibition.

Enzyme activity was determined by measuring the rate of turnover of theartificial substrate (A) at 37° C. in 50 mM HEPES(N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid) buffer containing150 mM NaCl, pH 7.4.

Assays were performed in 96 well titre plates.

Lp-PLA₂ was pre-incubated at 37° C. with vehicle or test compound for 10min in a total volume of 180 μl. The reaction was then initiated by theaddition of 20 μl 10× substrate (A) to give a final substrateconcentration of 20 μM. The reaction was followed at 405 nm for 20minutes using a plate reader with automatic mixing The rate of reactionwas measured as the rate of change of absorbance.

Results:

Com- pound No XR¹ R Z IC₅₀(μM) 1 O(CH₂)₁₅CH₃ CH₃CONH N⁺(CH₃)₃ 0.8 2O(CH₂)₁₇CH₃ CH₃CONH N⁺(CH₃)₃ 3.5 3 O(CH₂)₁₇CH₃ CH₃CONH S⁺(CH₃)2 1.0 4O(CH₂)₁₇CH₃ CH₃CONH SCH₃ 0.08 5 O(CH₂)₁₇CH₃ CH₃CONH OH 0.45 6O(CH₂)₁₇CH₃ CH₃CONH OAc 0.2 7 O(CH₂)₁₇CH₃ CH₃CONH

0.5 8 O(CH₂)₁₇CH₃ CH₃CONH

0.55 9 O(CH₂)₁₇CH₃ CF₃CONH N⁺(CH₃)₃ 2.52. Copper Stimulated LDL Oxidation:

Copper stimulated oxidation of LDL is routinely measured by followingthe increase in conjugated diene formation by monitoring the change inabsorption at 234 nm. This assay can be used to study inhibitors ofoxidative modification of LDL. FIG. 1 demonstrates that Lp-PLA₂inhibitors are effective inhibitors of LDL oxidation through aprolongation of the lag phase, using compound 4 as an example.

3. Inhibition of Cu²⁺ Stimulated Lyso-Phosphatidylcholine (Lyso-PtdCho)Formation.

A 1 ml aliquot of human LDL (0.25 mg protein/ml) was incubated for 15min at 37° C. with compound or vehicle. 5 μM Cu²⁺ was then added toallow oxidation/lyso-PtdCho formation to occur. The incubation wasterminated by the addition of 3.75 ml chloroform/methanol/c HCl(200:400:5,v/v/v). Following the addition of 125 ml chloroform and 1.25ml 0.1M HCl, the mixture was vortexed and centrifuged. The lower phasewas carefully removed and the upper phase re-extracted with an equalvolume of synthetic lower phase. The extracts were pooled and driedunder nitrogen.

Phospholipids were reconstituted into 50 μl chloroform-methanol (2:1v/v). 10 μl aliquots were spotted on to pre-run silica gel HPTLC platesand then developed in chloroform/methanol 25–30% methylamine (60:20:5v/v/v). Plates were subsequently sprayed with the flourescent indicator,2-p-toluidinylnaphthalene-6-sulphonic acid (1 mM in 50 mM Tris/HCl, pH7.4) to identify phospholipid components. Fluorescence was measured at222 nm using a CAMAG TLC scanner. Lipoprotein lyso-PtdCho content wasquantified using a standard curve (0.05–0.5 μg) prepared in parallel.

Compound 4 dose dependently inhibits LDL lyso-PtdCho accumulationstimulated by copper ions with an IC ₅₀ value of −30 μM

4. Purification of Lipoprotein Associated Lp-PLA₂

Low density lipoprotein (LDL) was obtained by plasma apheresis. The LDLwas dialysed against 0.5 M NaCl, 50 mM MES (4-morpholine ethanesulphonic acid), pH=6.0 overnight at 4° C. Solid CHAPS(3-[(-3-cholamidopropyl)dimethylamino]-1-propane sulphonate) was addedto 10 mM and the LDL stirred for 30 minutes to effect solubilisation.The solubilised LDL was pumped onto a pre-equilibrated Blue Sepharose6FF (Pharmacia) column (2.6×20 cm). The column was then washed with 50mM MES, 10 mM CHAPS, 0.5 M NaCl, pH=6.0 followed by 50 mM Tris, 10 mMCHAPS, 0.5 M NaCl, pH=8.0 until the absorbance (280 nm) of the eluatereached zero. Lp-PLA₂ was eluted using 50 mM Tris, 10 mM CHAPS, 1.5 MNaCl, pH=8.0. The Lp-PLA₂ fraction was then concentrated and dialysedovernight against 50 mM Tris, 10 mM CHAPS, pH=8.0.

The dialysed Lp-PLA₂ was submitted to anion exchange chromatography on amono Q column (Pharmacia) using 50 mM Tris, 10 mM CHAPS, pH=8.0 with aNaCl gradient from zero to 0.3 M. The Lp-PLA₂ fractions obtained fromthe mono Q column were applied directly to a Hi Trap Blue cartridge(Pharmacia). The cartridge was washed with 50 mM Tris, 10 mM CHAPS, 0.5M NaCl, pH=8.0 until the absorbance of the eluate (280 nm) was zero.Lp-PLA₂ was then eluted using 50 mM Tris, 10 mM CHAPS, 1.5 M NaCl,pH=8.0. This gave Lp-PLA₂ which is greater than 95% pure as shown inFIG. 2. This also demonstrates that the native enzyme is extensivelyglycosylated.

5. Enzyme Sequence

The purity of the final enzyme preparation was verified by fivecriteria 1) SDS-polyacrylamide gel electrophoresis gave one band forboth native and de-glycosylated forms. 2) Reverse phase high pressureliquid chromatography (RP-HPLC) gave a single peak, 3) The intactpreparation gave no results by protein sequencing, implying that theprotein was N-terminally blocked and free of any contaminants with openN-terminals, 4) By laser desorbtion mass spectometry only one broad peakwas observed with de-glycosylated protein, and 5) none of the sectionsof extended peptide data from sequencing gave any database matchesindicative of contaminating proteins. Three cleavage strategies wereused to obtain internal sequence information; trypsin (afterde-glycosylation), cyanogen bromide (methionine cleavage) andBNPS-Skatol (tryptophan cleavage). The resulting peptides were separatedby RP-HPLC, collected and sequenced. The accumulated sequence dataallowed several extended stretches of primary structure of the Lp-PLA2enzyme to be verified. These are shown below as Peptides 1, 2, 3 and 4(SEQ ID Nos 1 to 4). When searched against the National Centre forBiotechnological information (NCBI) non-redundant peptide sequencedatabases no high similarity matches were obtained. Estimation of themolecular weight of pure, de-glycosylated protein by laser desorptionmass spectometry gives values in the region of 45–47 kDa (separately 45kDa and 46–47 kDa), indicating that the sequences constituteapproximately 15 to 20% of the protein.

6. Gene Sequence

Three expressed sequence tags (ESTs) from human cDNA libraries have beenfound to have extensive alignments with the Peptide Sequences 1 to 3.These EST's are shown below as Nucleotide Sequences 1 to 3 (SEQ ID NOS:5 to 7) Nucleotide Sequence 1 is a 420 base sequence derived from ahuman foetal spleen library. Nucleotide Sequence 2 is a 379 basesequence derived from a 12-week human embryo library. NucleotideSequence 3 is a 279 base sequence derived from a T-cell lymphomalibrary. The identities at both the nucleic acid and amino acid leveljustified an overlapping alignment of the cDNA of all three ESTs,Nucleotide Sequences 3 (bases 1–278), I (bases 1–389) (in reverseorientation) and 2 (bases 1–304) with the Peptide Sequences 1, 2 and 3(partially). Beyond these limits, the poor resolution of the rawsequence data precludes accurate base calling.

There are two remaining unassigned peptide sections from Peptides 3 and4, both of which are expected to be present in the complete protein,-Q-Y-I-N-P-A-V-, and W-L-M-G-N-I-L-R-L-L-F-G-S-M-T-T-P-A-N-.

7. Isolation of Full-Length Lp-PLA₂ cDNA

The full DNA sequence was determined for the clone (HLTA145) from whichthe Lymphoma EST (SEQ ID NO: 7) was derived, giving a total of 572bases; SEQ ID NO: 8. There is one base difference between this sequenceand the EST (between bases 1 to 256 of the EST); at position 27 ofHLTA145 there is an A compared with a T in the EST. This would cause acoding change; L in HLTA145 compared with F in the EST. Clone HLTA145was used as a radiolabelled probe to screen the Lymphoma cDNA library inorder to isolate the full-length Lp-PLA₂ clone. The library was preparedin the bacteriophage λ vector, Unizap XR (Stratagene).

Preparation of the Filters for Screening

The library was plated out at a density of 20,000 plaques per 150 mmpetri dish onto E.coli XL-1 Blue host cells (ie. 200,000 plaques on 10dishes). An overnight of XL-1 Blue was prepared in 100 mls LB/0.2% w/vMaltose/10 mM MgSO₄. The cells were pelleted, resuspended in 50 mls 10 mmM MgSO₄ and stored on ice. 180 μl of the library bacteriophage stock(23,400 pfu's) were added to 7 mls XL-1 Blue cells, mixed and dividedinto 10 aliqouts of 615 μl. The 10 tubes were incubated at 37° C. for 30minutes. 7 mls of molten (@45° C.) top agarose (0.7% wv agarose in LB)were added, mixed well and poured onto 150 mm LB agar plates (1.5% w/vagar in LB). The plates were inverted and incubated at 37° C. forapproximately 7.5 hour. The plates were held at 4° C. until needed.

The plaques were transfered to 132 mm Hybond-N nylon filters (AmershamInternational) by laying the filters on the plates for 2 minutes (4minutes for the duplicate). The DNA's on the filters were denatured for2 minutes (0.5M NaCl, 1.5M NaOH), neutralised for 4 minutes (15M NaCl,1.0M Tris pH7.4) and the filters placed on 2×SSC for 1 minute. Thefilters were then dried and the DNA cross-linked to the filter using aStratalinker UV 2400 (Stratagene) at 120,000 μJoules/cm².

The filters were pre-hybridised in 1 mM EDTA, 0.5M NaHPO₄, 7% SDS(Church, G M. and Gilbert, W. (1984) PNAS USA 81 p1991–1995) in a TechneHB2 hybridisation oven at 55° C. for 3 hours. Each bottle contained 2filters and 25 mls prehybridization solution.

Preparation of the Radiolabelled Probe

The probe cDNA (from HLTA 145) was excised from pBluescript II SK+/− asan approximately 600 bp EcoRI-XhoI fragment and approximately 100 ng ofgel purified fragment were labelled using 1.2 MBq ³²P dATP and 1.2 MBq³²P dCTP by PCR labelling using Taq DNA polymerase (Boehringer Mannheim)and primers designed to prime at the 5′ and 3′ ends of the EST sequence.The labelling reaction was carried out in a total volume of 200 μl andincluded unlabelled dNTP's at the following concentrations:

-   -   dATP 20 μM    -   dCTP 20 μM    -   dGTP 200 μM    -   dTTP 200 μM        The PCR reaction was carried out over 35 cycles of:    -   94° C. for 30 s    -   60° C. for 30 s    -   72° C. for 30 s        Screening

The radiolabelled probe was denatured at 98° C. for 5 minutes anddivided into 10 aliquots of 20 μl. One aliquot was added perhybridisation bottle. Hybridisation was carried out over 16 hours at 55°C. The filters were washed at 60° C. (2×10 minutes) with 0.1% w/y SDS,0.1×SSC (50 mls per wash per bottle). The filters were autoradiographedand the films (Fuji Medical X-Ray Film) developed after 5 days exposure.

Duplicate positives were progressed to a secondary screen. The plaqueswere cored out into 1 ml SM (100 mM NaCl, 10 mM MgSO₄, 1M Tris, pH7.4),titrated and plated onto 90 mm petri dishes at between 20 and 200 pfu'sper dish. The secondary screen was carried out as described for theprimary screen except the filters were washed at 65° C. Theautoradiographs were developed after 16 hours exposure.

DNA Sequencing

The duplicated positive clones from the secondary screen were excisedfrom the λ Unizap XR bacteriophage vector into the Bluescript phagemid(according to the Stratagene manual) for characterisation. One of theclones, carrying an insert of approximately 1.5 kb, was sequenced onboth strands (using the USB Sequenase 2.0 DNA sequencing kit) by primerwalking (SEQ ID NO: 9). The cDNA has an open reading frame with thepotential to code for a polypeptide of 441 amino acids.

The 3′ region of the full-length cDNA aligns with the HLTA145 sequencewith the exception of 3 mismatches (see below). The predictedpolypeptide sequence of the lymphoma Lp-PLA₂ is shown as SEQ ID NO: 9.

Inspection of the full length cDNA (SEQ ID NO: 9) reveals probableerrors in Peptide 3. One of these errors is in the assignment ofcontinuity between V-M which is incompatible with the perfect sequencematch with the cDNA after this position. It seems likely that a shortpeptide, containing the sequence -Q-Y-I-N-P-, co-purified with thelonger cyanogen bromide partial cleavage peptide and, by being presentin greater quantity, was assigned as the major sequence and contiguouswith the subsequent amino acids. The remaining section of Peptide 3 andthe whole of Peptide 4 can be identified in the predicted full lengthenzyme sequence (SEQ ID No: 9). It thus seems likely that Peptide 3 isin fact two separate Peptides 5 (SEQ ID No:10) and 6 (SEQ ID NO: 11).The second probable error has occurred in the transcription from the rawdata for Peptide 3 which on checking was consistent with Peptide 5having the sequence -Q-Y-I-N-P-V-A, rather than Q-Y-I-N-P-A-V-.

The 3 base differences are as follows:

-   1) T at 859 is A in HLTA145; aminoacid change F in full-length, L in    HLTA145. (Note that the original EST is identical with the    full-length cDNA at position 859).-   2) C at 1173 is T in HLTA145; aminoacid change A in full-length, V    in HLTA145.-   3) T at 1203 is C in HLTA145; aminoacid change L in full-length, S    in HLTA145.

The peptide data and the Foetal Spleen EST sequence (SEQ ID NO: 5)support the full-length cDNA sequence for differences (2) and (3)although the Human Embryo EST (SEQ ID NO: 6) is identical to theLymphoma EST (SEQ ID No: 7) at position 1173. The Human Embryo EST (SEQID NO: 6) has a further difference (4) corresponding to position 1245 inthe full-length Lymphoma sequence (SEQ ID NO: 9)(comparison betweenbases 2 to 304 of the Human Embryo EST and the full-length LymphomacDNA).

-   4) A at 1245 is T in the Embryo EST (SEQ ID NO: 6)(amino acid change    D to V in the Embryo EST). Peptide data covering this region    supports the Lymphoma DNA sequence (SEQ ID NO: 9).

The Lp-PLA₂ DNA sequence from 848 to 1361 of SEQ ID NO: 9 (amino acidresidues 271 to 441 of SEQ ID NO: 9) is the region for which all majordata sets agree substantially, ie. the peptide data, the Foetal spleen,full-length Lymphoma and it includes the known active site and istherefore believed to be a significant characterising region for theLp-PLA₂ enzyme.

The predicted MW for the fill reading frame is 50090. This in in exessof that determined for the de-glycosylated, purified protein butpost-translational events could account for this discrepancy. The mostlikely of these are the removal of an N-terminal signal peptide and/orlimited proteolytic degradation of the protein C-terminal. The lattercould occur in-vivo, during purification, or under the conditions ofde-glycosylation.

Diagnostic Method

A sample of blood is taken from a patient, the plasma/serum sampleprepared and passed through a dextran sulphate column pre-equilibratedwith 0.9% (w\v) NaCl solution. Following washes with the same saltsolution Lp-PLA₂ is eluted with a 4.1% (w\v) NaCl solution. Heparinagarose columns can also be used with the wash and elution solutionscontaining 0.7% and 2.9% NaCl, respectively. Enzyme present in thesample is determined by assaying for either

-   (a) enzyme activity:

The substrate (A) (see structure in 1) is used to assay Lp-PLA₂ activityby monitoring the absorbance change at 400 nm. Purified enzyme ispre-incubated at 37° C. and substrate (50 μM) is added after 5 minutes.The absorbance change at 400 nm is monitored for 20-minutes. Thissubstrate has previously been reported as a substrate for classicalcalcium-dependent PLA₂s. (Washburn, W. N. and Dennis, E. A., J. AmerChem. Soc., 1990, 112, 2040–2041); or

-   (b) protein content

Total protein content (i.e. enzyme content) can be determined usingpolyclonal antiserum raised against purified human Lp-PLA₂. The antiserarecognises both native and glycosylated enzyme as measured byimmunoprecipitation of activity and Western Blot analysis.

Polyclonal antiserum was prepared as follows. Immunisation of rabbitsinvolved mixing 0.5 ml of purified human Lp-PLA₂ (=100 μg) with an equalvolume of Freund's complete adjuvant. The final emulsion was givensubcutaneously in 4×0.25 ml injections. Boosting using a Freund'sincomplete adjuvant antigen mixture (4×0.25 ml subcut; dosage=50 μg)took place 4 weeks later. Adequate titre was evident at between 6–8weeks from initial injection.

IN THE FIGURES

FIG. 1 is a graph of absorbance at 234 nm against time (min) in a studyof inhibition of copper (5 μM)- stimulated LDL (150 μg/ml) oxidation bycompound 4 vs control vehicle.

FIG. 2 is an analysis the purified Lp-PLA₂ material of Example 4following separation by polyacrylamide gel electrophoresis. Lanes 2, 4and 6 contain adjacent fractions of purified native Lp-PLA₂. Lanes 1, 3and 5 are fractions 2, 4 and 6 respectively after N-deglycosylation.

SEQUENCE DATA

SEQ. ID. No: 1 - Peptide 1-M-L-K-L-K-G-D-I-D-S-N-A-A-I-D-L-S-N-K-A-S-L-A-F-L-Q-K-H-L-G-L-H-K-D-F-D-Q- SEQ. ID. No: 2 - Peptide 2-W-M-F-P-L-G-D-E-V-Y-S-R-I-P-Q-P-L-F-F-I-N-S-E-Y-F-Q-Y- P-A-N- SEQ. ID.No: 3 - Peptide 3-Q-Y-I-N-P-A-V-M-I-T-I-R-G-S-V-H-Q-N-F-A-D-F-T-F-A-T-G- SEQ. ID. No: 4 -Peptide 4 -W-L-M-G-N-I-L-R-L-L-F-G-S-M-T-T-P-A-N SEQ. ID. No: 5 -Nucleotide Sequence 1 1 AAAAAACCTA TTTTAATCCT AATTGTATTT CTCTATTCCTGAAGAGTTCT 51 GTAACATGAT GTGTTGATTG GTTGTGTTAA TGTTGGTCCC TGGAATAAGA 101TTCTCATCAT CTCCTTCAAT CAAGCAGTCC CACTGATCAA AATCTTTATG 151 AAGTCCTAAATGCTTTTGTA AGAATGCTAA TGAAGCTTTG TTGCTAAGAT 201 CAATAGCTGC ATTTGAATCTATGTCTCCCT TTAATTTGAG CATGTGTCCA 251 ATTATTTTGC CAGTNGCAAA AGTGAAGTCAGCAAAATTCT GGTGGACTGA 301 ACCCCTGATT GTAATCATCT TTCTTTCTTT ATCAGGTGAGTAGCATTTTT 351 TCATTTTTAT GATATTAGCA GGATATTGGA AATATTCAGN GTTGNTAAAA401 AGNGGNGGCT GAGGGATTCT SEQ. ID. No: 6 - Nucleotide Sequence 2 1TGCTAATATC ATAAAAATGA AAAAATGCTA CTCACCTGAT AAAGAAAGAA 51 AGATGATTACAATCAGGGGT TCAGTCCACC AGANTTTTGC TGACTTCACT 101 TTTGCAACTG GCAAAATAATTGGACACATG CTCAAATTAA AGGGAGACAT 151 AGATTCAAAT GTAGCTATTG ATCTTAGCAACAAAGCTTCA TTAGCATTCT 201 TACAAAAGCA TTTAGGACTT CATAAAGATT TTGTTCAGTGGGACTGCTTG 251 ATTGAAGGAG ATGATGAGAA TCTTATTCCA GGGACCAACA TTAACACAAC301 CAATTCAACA CATCATGTTT ACAGAACTTC TTCCAGGGAA TAGGAGGAAA 351TACAATTGGG GTTTAAAATA GGTTTTTTT SEQ. ID. No: 7 - Nucleotide Sequence 3 1GAAGAATGCA TTAGATTTAA AGTTTGATAT GGAACAACTG AAGGACTCTA 51 TTGATAGGGAAAAAATAGCA GTAATTGGAC ATTCTTTTGG TGGAGCAACG 101 GTTATTCAGA CTCTTAGTGAAGATCAGAGA TTCAGATGTG GTATTGCCCT 151 GGATGCATGG ATGTTTCCAC TGGGTGATGAAGTATATTCC AGAATTCCTC 201 AGCCCCTCTT TTTTATCAAC TCTGAATATT TCCAATATCCTGCTAATATC 251 ATAAAANTGG AAAAATGCTA CTCACCTGG Seq. ID. No:8 - DNAsequence of HLTA145         10         20         30         40        50 AAAATAGCAG TAATTGGACA TTCTTTAGGT GGAGCAACGG TTATTCAGAC        60         70         80         90        100 TCTTAGTGAAGATCAGAGAT TCAGATGTGG TATTGCCCTG GATGCATGGA        110        120       130        140        150 TGTTTCCACT GGGTGATGAA GTATATTCCAGAATTCCTCA GCCCCTCTTT        160        170        180        190       200 TTTATCAACT CTGAATATTT CCAATATCCT GCTAATATCA TAAAAATGAA       210        220        230        240        250 AAAATGCTACTCACCTGATA AAGAAAGAAA GATGATTACA ATCAGGGGTT        260        270       280        290        300 CAGTCCACCA GAATTTTGCT GACTTCACTTTTGCAACTGG CAAAATAATT        310        320        330        340       350 GGACACATGC TCAAATTAAA GGGAGACATA GATTCAAATG TAGCTATTGA       360        370        380        390        400 TCTTAGCAACAAAGCTTCAT CAGCATTCTT ACAAAAGCAT TTAGGACTTC        410        420       430        440        450 ATAAAGATTT TGATCAGTGG GACTGCTTGATTGAAGGAGA TGATGAGAAT        460        470        480        490       500 CTTATTCCAG GGACCAACAT TAACACAACC AATCAACACA TCATGTTACA       510        520        530        540        550 GAACTCTTCAGGAATAGAGA AATACAATTA GGATTAAAAT AGGTTTTTTA        560        570AAAAAAAAAA AAAAAAAACT CG SEQ. ID. No:9 - cDNA Sequence of LymphomaLp-PLA₂         10        20        30        40        50TGAGAGACTAAGCTGAAACTGCTGCTCAGCTCCCAAGATGGTGCCACCCA                                     M  V  P  P  K        60        70        80        90       100AATTGCATGTGCTTTTCTGCCTCTGCGGCTGCCTGGCTGTGGTTTATCCT  L  H  V  L  F  C  L  C  G  C  L  A  V  V  Y  P       110       120       130       140       150TTTGACTGGCAATACATAAATCCTGTTGCCCATATGAAATCATCAGCATGF  D  W  Q  Y  I  N  P  V  A  H  M  K  S  S  A  W       160       170       180       190       200GGTCAACAAAATACAAGTACTGATGGCTGCTGCAAGCTTTGGCCAAACTA V  N  K  I  Q  V  L  M  A  A  A  S  F  G  Q  T  K       210       220       230       240       250AAATCCCCCGGGGAAATGGGCCTTATTCCGTTGGTTGTACAGACTTAATG  I  P  R  G  N  G  P  Y  S  V  G  C  T  D  L  M       260       270       280       290       300TTTGATCACACTAATAAGGGCACCTTCTTGCGTTTATATTATCCATCCCAF  D  H  T  N  K  G  T  F  L  R  L  Y  Y  P  S  Q       310       320       330       340       350AGATAATGATCGCCTTGACACCCTTTGGATCCCAAATAAAGAATATTTTT D  N  D  R  L  D  T  L  W  I  P  N  K  E  Y  F  W       360       370       380       390       400GGGGTCTTAGCAAATTTCTTGGAACACACTGGCTTATGGGCAACATTTTG  G  L  S  K  F  L  G  T  H  W  L  M  G  N  I  L       410       420       430       440       450AGGTTACTCTTTGGTTCAATGACAACTCCTGCAAACTGGAATTCCCCTCTR  L  L  F  G  S  M  T  T  P  A  N  W  N  S  P  L       460       470       480       490       500GAGGCCTGGTGAAAAATATCCACTTGTTGTTTTTTCTCATGGTCTTGGGG R  P  G  E  K  Y  P  L  V  V  F  S  H  G  L  G  A       510       520       530       540       550CATTCAGGACACTTTATTCTGCTATTGGCATTGACCTGGCATCTCATGGG  F  R  T  L  Y  S  A  I  G  I  D  L  A  S  H  G       560       570       580       590       600TTTATAGTTGCTGCTGTAGAACACAGAGATAGATCTGCATCTGCAACTTAF  I  V  A  A  V  E  H  R  D  R  S  A  S  A  T  Y       610       620       630       640       650CTATTTCAAGGACCAATCTGCTGCAGAAATAGGGGACAAGTCTTGGCTCT Y  F  K  D  Q  S  A  A  E  I  G  D  K  S  W  L  Y       660       670       680       690       700ACCTTAGAACCCTGAAACAAGAGGAGGAGACACATATACGAAATGAGCAG  L  R  T  L  K  Q  E  E  E  T  H  I  R  N  E  Q       710       720       730       740       750GTACGGCAAAGAGCAAAAGAATGTTCCCAAGCTCTCAGTCTGATTCTTGAV  R  Q  R  A  K  E  C  S  Q  A  L  S  L  I  L  D       760       770       780       790       800CATTGATCATGGAAAGCCAGTGAAGAATGCATTAGATTTAAAGTTTGATA I  D  H  G  K  P  V  K  N  A  L  D  L  K  F  D  M       810       820       830       840       850TGGAACAACTGAAGGACTCTATTGATAGGGAAAAAATAGCAGTAATTGGA  E  Q  L  K  D  S  I  D  R  E  K  I  A  V  I  G       860       870       880       890       900CATTCTTTTGGTGGAGCAACGGTTATTCAGACTCTTAGTGAAGATCAGAGH  S  F  G  G  A  T  V  I  Q  T  L  S  E  D  Q  R       910       920       930       940       950ATTCAGATGTGGTATTGCCCTGGATGCATGGATGTTTCCACTGGGTGATG F  R  C  G  I  A  L  D  A  W  M  F  P  L  G  D  E       960       970       980       990      1000AAGTATATTCCAGAATTCCTCAGCCCCTCTTTTTTATCAACTCTGAATAT  V  Y  S  R  I  P  Q  P  L  F  F  I  N  S  E  Y      1010      1020      1030      1040      1050TTCCAATATCCTGCTAATATCATAAAAATGAAAAAATGCTACTCACCTGAF  Q  Y  P  A  N  I  I  K  M  K  K  C  Y  S  P  D      1060      1070      1080      1090      1100TAAAGAAAGAAAGATGATTACAATCAGGGGTTCAGTCCACCAGAATTTTG K  E  R  K  M  I  T  I  R  G  S  V  H  Q  N  F  A      1110      1120      1130      1140      1150CTGACTTCACTTTTGCAACTGGCAAAATAATTGGACACATGCTCAAATTA  D  F  T  F  A  T  G  K  I  I  G  H  M  L  K  L      1160      1170      1180      1190      1200AAGGGAGACATAGATTCAAATGCAGCTATTGATCTTAGCAACAAAGCTTCK  G  D  I  D  S  N  A  A  I  D  L  S  N  K  A  S      1210      1220      1230      1240      1250ATTAGCATTCTTACAAAAGCATTTAGGACTTCATAAAGATTTTGATCAGTL  A  F  L  Q  K  H  L  G  L  H  K  D  F  D  Q  W      1260      1270      1280      1290      1300GGGACTGCTTGATTGAAGGAGATGATGAGAATCTTATTCCAGGGACCAAC  D  C  L  I  E  G  D  D  E  N  L  I  P  G  T  N      1310      1320      1330      1340      1350ATTAACACAACCAATCAACACATCATGTTACAGAACTCTTCAGGAATAGAI  N  T  T  N  Q  H  I  M  L  Q  N  S  S  G  I  E       1360 GAAATACAATT K  Y  N SEQ. ID. No: 10 - Peptide 5 -Q-Y-I-N-P-V-A- SEQ. ID. No: 11 -Peptide 6 -M-I-T-I-R-G-S-V-H-Q-N-F-A-D-F-T-F-A-T-G-

1. A method of diagnosing a patients susceptibility to atheroscleroticdisease which comprises assaying a sample from a patient for Lp-PLA₂enzyme activity wherein elevated Lp-PLA₂ enzyme activity in the sampleof the patient as compared to a standard is indicative of the patient'ssusceptibility to atherosclerotic disease.
 2. The method according toclaim 1 wherein the atherosclerotic disease is selected from the groupcomprising atherosclerosis, stroke and myocardial infarction.
 3. Themethod according to claim 1 wherein the atherosclerotic disease isatherosclerosis.
 4. The method according to claim 1 wherein theatherosclerotic disease is stroke.
 5. The method according to claim 1wherein the atherosclerotic disease is myocardial infarction.
 6. Themethod according to claim 1 wherein the sample is selected from thegroup comprising blood, serum and plasma.
 7. The method according toclaim 1 wherein the sample is blood.
 8. The method according to claim 1wherein the sample is serum.
 9. The method according to claim 1 whereinthe sample is plasma.
 10. The method according to claim 1 wherein thesample is assayed in a 96 well titre plate.
 11. The method of accordingto claim 1 wherein the sample is assayed for enzymatic activity via anabsorbance assay.
 12. The method according to claim 11 whereinabsorbance at 400 nm or 405 nm is monitored.
 13. The method of accordingto claim 1 wherein the sample is assayed for enzymatic activity via anabsorbance assay measuring rate of turnover of an artificial substrate.14. The method according to claim 13 wherein absorbance at 400 nm or 405nm is monitored.
 15. The method according to claim 13 wherein theartificial substrate comprises a nitrophenyl group.
 16. A method ofdiagnosis of a patient's susceptibility to atherosclerotic disease whichcomprises analyzing a sample from a patient for the presence of theenzyme Lp-PLA₂ by assaying the sample for enzyme activity via anabsorbance assay measuring rate of turnover of an artificial substratewherein elevated Lp-PLA₂ enzyme activity in the sample of the patient ascompared to a standard are indicative of the patient's susceptibility toatherosclerotic disease.
 17. The method of claim 16 wherein theartificial substrate comprises a nitrophenyl group.
 18. A methodaccording to claim 16 wherein the disease is atherosclerosis, stroke ormyocardial infarction.
 19. The method according to claim 16 wherein thesample is selected from the group consisting of blood, serum, andplasma.
 20. The method according to claim 16 wherein the sample isblood.
 21. The method according to claim 16 wherein the sample isplasma.
 22. The method according to claim 16 wherein the sample isserum.
 23. The method according to claim 16 wherein the disease isatherosclerosis.
 24. The method according to claim 16 wherein thedisease is stroke.
 25. The method according to claim 16 wherein thedisease is myocardial infarction.
 26. The method according to claim 16wherein the absorbance assay is performed in a 96 well titre plate. 27.The method according to claim 16 wherein absorbance at 400 nm or 405 nmis monitored.